Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):
- D01 C
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D02 C
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D19 C
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D20 C
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T01 C
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T02 C
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T19 C
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T20 C
RNA was isolated using RNAzol RT (Molecular Research Center) in the following way:
- Unweighed, frozen tissue transferred to 500uL of RNAzol RT and immediately homogenized with disposable pestle.
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Added additional 500uL of RNAzol RT and vortexed to mix.
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Added 400uL of 0.1% DEPC-treated H2O, vortexed and incubated 15mins at RT.
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Centrifuged 12,000g for 15mins at RT.
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Transferred 750uL of supernatant to clean tube (discarded remainder), added 1 volume (750uL) of isopropanol, vortexed, and incubated at RT for 10mins.
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Centrifuged 12,000g for 10mins at RT.
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Discarded supernatant.
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Washed pellet with 75% ethanol (made with 0.1% DEPC-treated H2O).
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Centrifuged 4,000g for 2mins at RT.
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Discarded supernatant and repeated wash steps.
Pellet was resuspended in 50uL of 0.1% DEPC-treated H2O and stored @ -80oC in Ronit’s temporary box.
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